Non-heme iron-sulfur centers are of widespread importance in many aspects of biology and medicine. This proposal focuses on two of the most challenging enzymes in this group: the Ni-Fe hydrogenases and the Fe-only hydrogenases. Recent crystallographic and spectroscopic studies have dramatically altered the perceived roles of the metal centers in these enzymes. Significant new roles for the iron centers at the active sites of these enzymes are proposed. The active site of Ni-Fe hydrogenase contains an unexpected and unprecedented [Fe(CN)2(CO)] center. Fe-only hydrogenases contains a six Fe center with a unique binuclear assembly: Fe2(CN)2(CO)2(1,3- propanedithiolate). These are the first instances that either CN- or CO has been found as a native ligand in a metalloprotein. Synthetic analogs of certain aspects of the active sites of these enzymes will be prepared and characterized with respect to their structural, spectroscopic and reactivity properties. Model studies suggest that the [Fe(CN)2(CO)] unit in Ni-Fe hydrogenase remains low-spin Fe(II) during the enzymatic reaction. A hypothesis, that the reactivity of Ni-Fe hydrogenase occurs at the [Fe(CO)(CN)2] center and at a [Fe(CO)(CN)] center in Fe-only hydrogenases, will be tested. In these studies, the synthetic chemistry of Fe with CN- and CO ligands will be developed. The synthetic, spectroscopic and reactivity studies will assist in the determination of the mechanism by which these enzymes catalyze the activation and/or evolution of H2. From these studies, important new information will be added to the knowledge base of the bioinorganic chemistry of iron.